A continuous, farm-scale, solid-phase fermentation process for fuel ethanol and protein feed production from fodder beets

Fuel ethanol (95%) was produced from fodder beets in two farm-scale processes. In the first process, involving conventional submerged fermentation of the fodder beets in a mash, ethanol and a feed (PF) rich in protein, fat, and fiber were produced. Ethanol yields of 70 L/metric ton (7 gal/ton) were obtained; however, resulting beers had low ethanol concentrations [3-5% (v/v)]. The high viscosity of medium and low sugar, beet mashes caused mixing problems which prevented any further increase of beet sugar in the mash. The severely limited the maximum attainable ethanol concentration during fermentation, thereby making the beer costly to distill into fuel ethanol and the process energy inefficient. In order to achieve distillably worthwhile ethanol concentrations of 8-10% (v/v), we developed and tested a solid-phase fermentation process (continuous). In preliminary trials, this system produced fermented pulp with over 8% (v/v) ethanol corresponding to an ethanol yield of 87 L/metric ton (21 gal/ton). Production costs with this novel process are $0.47/L ($1.77/gal) and the energy balance is 2.11. These preliminary cost estimates indicate that fodder beets are potentially competitive with corn as an ethanol feedstock. Additional research, however, is warranted to more precisely refine individual costs, energy balances and the actual value of the PF.     

Farm-scale production of fuel ethanol and wet grain from corn in a batch process

The batch production of fuel grade ethanol and distillers' wet grain (wet solids) in a farm-scale process (1240-15,580 L/batch) is described. The employs yeast fermentation of amylase-treated corn mash and a two-stage distillation. Primary emphasis in this study was on the cooking, fermentation, and centrifugation steps. Without recycling, fermentation of the mash yield beers with 10.0-10.5% ethanol. Recycling of stillage supernatant at full, 75, or 50% strengths produced enriched mashes that after 48-h fermentation yielded beers with 5-;14% more ethanol. Recycling twice with full-strength supernatant at pH 7.0 increased the ethanol yield in the final beer 16.5%; however, the time to complete the final fermentation was extended form 48 to 72 h and salt buildup occurred. By recycling at pH 5.4, it was possible to avoid rapids salt buildup and obtain beers with 10.3-10.5% ethanol. Recycling resulted in increased levels of glucose, starch, crude protein, and fat in the beer and a reduced moisture content while the wet solids showed an increased starch content. Centrifugation after cooking or fermentation yield in the subsequently produced beer. Fermentation of a volume-resorted mash supernatant gave a beer with only 9.25% ethanol. Mash wet solids varied somewhat chemically from beer and stillage solids. An economic and energy balance analysis of various modes of plant operation are provided and plant considerations are suggested.     

利用葡萄糖废母液生产酒精的研究

选育出两株利用葡萄糖废母液生产酒精的菌株S_(995)和S_8。S_(095)用于70％母液和30％糖蜜混合连续酒精发酵,醪液中酒精份平均可达10.1％。S_8用于50％、70％母液和50％、30％玉米糖化醪混合生产酒精,醪液中酒精份可达12.37％(实验室数据)和10％(实际生产数据)。     

Fuel ethanol and high protein feed from corn and corn-whey mixtures in a farm-scale plant

Distiller's wet grain (DWG) and 95% ethanol were produced from corn in a farm-scale process involving batch cooking-fermentation and continuous distillation-centrifugation. The energy balance was 2.26 and the cost was $1.86/gal (1981 cost). To improve the energy balance and reduce costs, various modifications were made in the plant. The first change, back-end (after liquefaction) serial recycling of stillage supernatant at 20 and 40% strengths, produced beers with 0.2 and 0.4% (v/v) more ethanol, respectively, than without recycling. This increased the energy balance by 0.22-0.43 units and reduced costs by $0.07-$0.10/gal. The DWGs from back-end recycling had increased fat. The second change, increasing the starch content from 17-19% to 27.5%, increased the ethanol in the beer from 10.5-14.9% at a cost saving of $0.41/gal. The energy balance increased by 1.08 units. No significant change was seen in DWG composition. The third change, using continuous cascade rather than batch fermentation, permitted batch-levels of ethanol (10%) in the beer but only at low dilution rates. Both the cost and energy balance were decreased slightly. The DWG composition remained constant. The last change, replacing part of the corn and all of the tap water in the mash with whole whey and using Kluyveromyces fragilis instead of Saccharomyces cerevisiae during fermentation, resulted in an energy balance increase of 0.16 units and a $0.27/gal cost reduction. Here, 10% ethanolic beers were produced and the DWGs showed increased protein and fat. Recommendations for farm-scale plants are provided.     

Biochemical oxygen demands of potential cellulose ethanol waste streams

Summary The effects of potential waste streams resulting from ethanol production by the simultaneous saccharification fermentation (SSF) of cellulose were determined by measuring the biochemical oxygen demand (BOD). A worst-case analysis of BOD from ethanol-containing SSF beer showed an initial value of 1670 mg/l BOD, which is 29% of the expected initial BOD. When ethanol was reduced to 0.1–0.2% w/v, BOD levels were 605 mg/l in the mash and 250 mg/l in the beer. Both values were well below the projected discharge levels.     

Lipids-free waxy corn starch as a substrate for distillery yeasts <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Utilization of lipids-free waxy starch by distillery yeasts in fuel ethanol production can contribute to better management of renewable resources, like cereals, especially maize Zea mays L. But the efficient conversion of starch into glucose-rich fermentable substrate, and subsequently to ethanol, needs more research on hydrolysis and fermentation conditions. The aim of this study was to evaluate the lack of natural corn grain lipids on the process of simultaneous saccharification and fermentation using chemometric techniques of designed experiments, commercial enzymatic preparations and distillery yeasts Saccharomyces cerevisiae CCY-11-3. Based on the results and statistical software support we can conclude that extraction of lipids from corn grains did not lead to statistically significant increase or decrease of glucose concentration in starch hydrolysis. The ethanol concentration in fermentation mash according to analysis was not statistically significantly affected by lipids extraction. The separated lipids could serve as a source of very valuable corn oil.     

Direct concentration and viability measurement of yeast in corn mash using a novel imaging cytometry method

Worldwide awareness of fossil-fuel depletion and global warming has been increasing over the last 30 years. Numerous countries, including the USA and Brazil, have introduced large-scale industrial fermentation facilities for bioethanol, biobutanol, or biodiesel production. Most of these biofuel facilities perform fermentation using standard baker’s yeasts that ferment sugar present in corn mash, sugar cane, or other glucose media. In research and development in the biofuel industry, selection of yeast strains (for higher ethanol tolerance) and fermentation conditions (yeast concentration, temperature, pH, nutrients, etc.) can be studied to optimize fermentation performance. Yeast viability measurement is needed to identify higher ethanol-tolerant yeast strains, which may prolong the fermentation cycle and increase biofuel output. In addition, yeast concentration may be optimized to improve fermentation performance. Therefore, it is important to develop a simple method for concentration and viability measurement of fermenting yeast. In this work, we demonstrate an imaging cytometry method for concentration and viability measurements of yeast in corn mash directly from operating fermenters. It employs an automated cell counter, a dilution buffer, and staining solution from Nexcelom Bioscience to perform enumeration. The proposed method enables specific fluorescence detection of viable and nonviable yeasts, which can generate precise results for concentration and viability of yeast in corn mash. This method can provide an essential tool for research and development in the biofuel industry and may be incorporated into manufacturing to monitor yeast concentration and viability efficiently during the fermentation process.     

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.     

Fermentation of sweet whey by ethanologenic Escherichia coli

Whey, an abundant byproduct of the dairy industry, contains large amounts of protein and lactose which could be used for fuel ethanol production. We have investigated a new organism as a candidate for such fermentations: recombinant Escherichia coli containing the genes encoding the ethanol pathway from Zymomonas mobilis. The highest level of ethanol achieved, 68 g/L, was produced after 108 hours in Luria broth containing 140 g lactose/L. Fermentations of lower lactose concentrations were completed more rapidly with approximately 88% of theoretical yields. Reconstituted sweet whey (60 g lactose/L)was fermented more slowly than lactose in Luria broth requiring 144 hours to produce 26 g ethanol/L. Supplementing sweet whey with a trace metal mix and ammonium sulfate reduced the required fermentation time to 72 hours and increased final ethanol concentration (28 g ethanol/L). By adding proteinases during fermentation, the requirement for ammonia was completely eliminated, and the rate of fermentation further improved (30 g ethanol/L after 48 hours). This latter incresed in rate of ethanol production and ethanol yield are presumed to result from incorporation of amino acids released by hydrolysis of whey proteins. The fermentation of sweet whey by ethanologenic E. coil reduced the nonvolatile residue by approximately 70%. This should reduce biological oxygen demand and reduce the cost of waste treatment. Whey supplemented with trace metals and small amounts of proteinase may represent an economically attractive feedstock for the production of ethanol and other useful chemicals.     

Interaction effects of lactic acid and acetic acid at different temperatures on ethanol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in corn mash

The combined effects of lactic acid and acetic acid on ethanol production by S. cerevisiae in corn mash, as influenced by temperature, were examined. Duplicate full factorial experiments (three lactic acid concentrations × three acetic acid concentrations) were performed to evaluate the interaction between lactic and acetic acids on the ethanol production of yeast at each of the three temperatures, 30, 34, and 37°C. Corn mash at 30% dry solids adjusted to pH 4 after lactic and acetic acid addition was used as the substrate. Ethanol production rates and final ethanol concentrations decreased (P<0.001) progressively as the concentration of combined lactic and acetic acids in the corn mash increased and the temperature was raised from 30 to 37°C. At 30°C, essentially no ethanol was produced after 96 h when 0.5% w/v acetic acid was present in the mash (with 0.5, 2, and 4% w/v lactic acid). At 34 and 37°C, the final concentrations of ethanol produced by the yeast were noticeably reduced by the presence of 0.3% w/v acetic acid and ≥2% w/v lactic acid. It can be concluded that, as in previous studies with defined media, lactic acid and acetic acid act synergistically to reduce ethanol production by yeast in corn mash. In addition, the inhibitory effects of combined lactic and acetic acid in corn mash were more apparent at elevated temperatures.     

Dry-grind process for fuel ethanol by continuous fermentation and stripping

Conversion of a high-solids saccharified corn mash to ethanol by continuous fermentation and stripping was successfully demonstrated in a pilot plant consuming 25 kg of corn per day. A mathematical model based on previous pilot plant results accurately predicts the specific growth rate obtained from these latest results. This model was incorporated into a simulation of a complete dry-grind corn-to-ethanol plant, and the cost of ethanol production was compared with that of a conventional process. The results indicate a savings of $0.03 per gallon of ethanol produced by the stripping process. The savings with stripping result from the capacity to ferment a more concentrated corn mash so there is less water to remove downstream.     

分批补料对木糖发酵生产乙醇的影响

以树干毕赤酵母为发酵菌种,纯木糖为发酵底物,通过分批补料来提高糖利用率以及乙醇得率。结果表明,在24h内,最佳初始木糖浓度为80g/L,在28h的发酵周期中,可以将木糖浓度提高至90g/L,在32h发酵周期内可以将木糖浓度提高至100g/L。通过分批补料,乙醇浓度得到明显提高。当总糖浓度分别为80g/L、90g/L时,24h发酵周期内,分批补料次数以1次为宜,乙醇浓度分别达30.95g/L、32.60g/L,相比于不补料即一次性投料,乙醇浓度分别提高了9.36%、9.18%。总糖浓度100g/L,28h发酵周期内,补料2次效果最佳,乙醇浓度达37.49g/L,比一次性投料下提高了10.36%,较一次性投料达到相同发酵效果缩短了4h。     

Improvements in Biobutanol Fermentation and Their Impacts on Distillation Energy Consumption and Wastewater Generation

In the conventional fermentation process to obtain butanol (a novel biofuel), product-induced toxicity results in a product stream with low concentration of butanol (～13 g/L) and limits the concentration of the sugar solution to less than 60 g/L. As a result, steam-consuming operations such as mash sterilization, downstream product recovery (distillation), and wastewater treatment are energy-intensive and important economic drawbacks. Based on the correlation between energy consumption of the distillation unit and butanol concentration in the fermentation beer, the present research points out that improvements in biobutanol processing intended to increase the concentration of butanol in the beer should have a minimum target of 36 g/L. Moreover, due to the dramatic effect of butanol concentration on the wastewater footprint, the volume of the effluent stream can be reduced by 60% (from 72 to 29 L stillage/L butanol) if the minimum concentration target is reached instead of the usual butanol titer of 13 g/L. These correlations were used as the basis to discuss the impacts of today’s research works (genetic strain improvement, utilization of lignocellulosic biomass feedstock, and development of new process technologies) on the energy consumption for complete dehydration of butanol and on wastewater generation.     

Low temperature pre-treatment of new cultivar of corn for ethanol production and nutrient value of its distiller’s dried grains with soluble

The objective of this work was to evaluate the production of bioethanol from a new Korean variety of corn (Gangdaok) and to assess low temperature pre-treatment of corn mashes before simultaneous saccharification and fermentation. Corn mashes containing 178 g/L of total sugar were fermented with Saccharomyces cerevisiae CHY 1011(KCTC 11250BP) at 35°C. Fermentation of mash supplemented with solid glucoamylase was completed within 48 h, and the ethanol produced was 474.0 and 473.1 L/ton as dry base with low temperature pre-treatment and pressure pretreatment, respectively. Furthermore, the DDGS of Gangdaok cultivar contained more essential amino acids (21.1 mg/g) than did Ambrosia cultivar (USA corn), which is a widely used feedstock. In addition, there were no significant differences in ethanol yield or amino acid concentration in DDGS between low temperature pre-treatment and pressure pretreatment. The results show that Gangdaok holds potential economic advantages if applied to the bioethanol and feed industries.     

Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation

External nutrient supplementation and detoxification of hydrolysate significantly increase the production cost of cellulosic ethanol. In this study, we investigated the feasibility of fermenting cellulosic hydrolysates without washing, detoxification or external nutrient supplementation using ethanologens Escherichia coli KO11 and the adapted strain ML01 at low initial cell density (16 mg dry weight/L). The cellulosic hydrolysates were derived from enzymatically digested ammonia fiber expansion (AFEX)-treated corn stover and dry distiller's grain and solubles (DDGS) at high solids loading (18% by weight). The adaptation was achieved through selective evolution of KO11 on hydrolysate from AFEX-treated corn stover. All cellulosic hydrolysates tested (36-52 g/L glucose) were fermentable. Regardless of strains, metabolic ethanol yields were near the theoretical limit (0.51 g ethanol/g consumed sugar). Volumetric ethanol productivity of 1.2 g/h/L was achieved in fermentation on DDGS hydrolysate and DDGS improved the fermentability of hydrolysate from corn stover. However, enzymatic hydrolysis and xylose utilization during fermentation were the bottlenecks for ethanol production from corn stover at these experimental conditions. In conclusion, fermentation under the baseline conditions was feasible. Utilization of nutrient-rich feedstocks such as DDGS in fermentation can replace expensive media supplementation.     

Potential of the waste from beer fermentation broth for bio-ethanol production without any additional enzyme, microbial cells and carbohydrates

The potential of the waste from beer fermentation broth (WBFB) for the production of bio-ethanol using a simultaneous saccharification and fermentation process without any extra additions of saccharification enzymes, microbial cells or carbohydrate was tested. The major microbial cells in WBFB were isolated and identified. The variations in compositions of WBFB with stock time were investigated. There was residual activity of starch hydrolyzing enzymes in WBFB. The effects of reaction modes e.g. static and shaking on bio-ethanol production were studied. After 7 days of cultivation using the supernatant of WBFB at 30 °C the ethanol concentration reached 103.8 g/L in shaking culture and 91.5 g/L in static culture. Agitation experiments conducted at a temperature-profile process in which temperature was increased from 25 to 67 °C shortened the simultaneous process time. The original WBFB was more useful than the supernatant of WBFB in getting the higher concentration of ethanol and reducing the fermentation time. From this whole study it was found that WBFB is a cheap and suitable source for bio-ethanol production.     

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol

In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195 degrees C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50 degrees C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30 degrees C. The phenols (0.4-0.5 g/L) and carboxylic acids (4.6-5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2.     

Pipeline transport and simultaneous saccharification of corn stover

Pipeline transport of corn stover delivered by truck from the field is evaluated against a range of truck transport costs. Corn stover transported by pipeline at 20% solids concentration (wet basis) or higher could directly enter an ethanol fermentation plant, and hence the investment in the pipeline inlet end processing facilities displaces comparable investment in the plant. At 20% solids, pipeline transport of corn stover costs less than trucking at capacities in excess of 1.4 M drytonnes/yr when compared to a mid range of truck transport cost (excluding any credit for economies of scale achieved in the ethanol fermentation plant from larger scale due to multiple pipelines). Pipelining of corn stover gives the opportunity to conduct simultaneous transport and saccharification (STS). If current enzymes are used, this would require elevated temperature. Heating of the slurry for STS, which in a fermentation plant is achieved from waste heat, is a significant cost element (more than 5 cents/l of ethanol) if done at the pipeline inlet unless waste heat is available, for example from an electric power plant located adjacent to the pipeline inlet. Heat loss in a 1.26 m pipeline carrying 2 M drytonnes/yr is about 5 degrees C at a distance of 400 km in typical prairie clay soils, and would not likely require insulation; smaller pipelines or different soil conditions might require insulation for STS. Saccharification in the pipeline would reduce the need for investment in the fermentation plant, saving about 0.2 cents/l of ethanol. Transport of corn stover in multiple pipelines offers the opportunity to develop a large ethanol fermentation plant, avoiding some of the diseconomies of scale that arise from smaller plants whose capacities are limited by issues of truck congestion.     

Gamma Irradiation on Fermentation Mashes Consisting Mainly of Cane Molasses

The application of the radiopasteurization method to fermentation media consisting mainly of molasses was investigated. γ-Irradiation was found to have an excellent pasteurization effect on the fermentation media and at the same time to bring about an increase in the fermentation rate and yield of ethanol. Percent survivals in molasses decreased to ca. 70% by heating at 80°C for 30 min, to ca. 10% by irradiation with 3.0 × 10⁵ rad and to ca. 1% by 6.0 × 10⁵ rad. Irradiated mash was suitable for the medium of the “starter”, since the rate and the degree of the growth of Saccharomyces cerevisiae in irradiated mash did not differ from those of the growth in heat-pasteurized mash.

In the case of the molasses mash supplemented with nitrogen sources, the fermentation rate and yield of ethanol in irradiated mash were larger than those in heated mash. Besides, in the absence of nitrogen sources a 14% difference in fermentation yield was seen between the mash irradiated with 3.0 × 10⁵ rad and the mash heated at 80°C. With the doses ranging from 1.0 × 10⁵ to 9.5 × 10⁵ rad, concentrations of total sugar and direct reducing sugar, pH, and optical density of molasses were little affected by irradiation.     

Real‐time monitoring of high‐gravity corn mash fermentation using in situ raman spectroscopy

In situ Raman spectroscopy was employed for real‐time monitoring of simultaneous saccharification and fermentation (SSF) of corn mash by an industrial strain of Saccharomyces cerevisiae. An accurate univariate calibration model for ethanol was developed based on the very strong 883 cm^?1 C–C stretching band. Multivariate partial least squares (PLS) calibration models for total starch, dextrins, maltotriose, maltose, glucose, and ethanol were developed using data from eight batch fermentations and validated using predictions for a separate batch. The starch, ethanol, and dextrins models showed significant prediction improvement when the calibration data were divided into separate high‐ and low‐concentration sets. Collinearity between the ethanol and starch models was avoided by excluding regions containing strong ethanol peaks from the starch model and, conversely, excluding regions containing strong saccharide peaks from the ethanol model. The two‐set calibration models for starch (R² = 0.998, percent error = 2.5%) and ethanol (R² = 0.999, percent error = 2.1%) provide more accurate predictions than any previously published spectroscopic models. Glucose, maltose, and maltotriose are modeled to accuracy comparable to previous work on less complex fermentation processes. Our results demonstrate that Raman spectroscopy is capable of real time in situ monitoring of a complex industrial biomass fermentation. To our knowledge, this is the first PLS‐based chemometric modeling of corn mash fermentation under typical industrial conditions, and the first Raman‐based monitoring of a fermentation process with glucose, oligosaccharides and polysaccharides present. Biotechnol. Bioeng. 2013; 110: 1654–1662. © 2013 Wiley Periodicals, Inc.